Ribonucleic acid (RNA) molecules are chains of nucleotides, each nucleotide containing a ribose, a nucleoside base, and a phosphate group. Messenger RNA (mRNA) plays a central role in the coding of genetic information and in converting the genetic code into proteins that carry out essential cellular functions. Thus, assays for the analysis of RNA molecules within cell or tissue samples are widely used by biologists to understand the molecular underpinnings of life. One of the challenges of these assays is the relative instability of RNA molecules leading to degradation. Assays to determine the quality of RNA samples can depend on a ratiometric determination of 28S to 18S RNA—however, such determinations only provide an approximation of RNA degradation status. Methods that provide accurate measurements of RNA degradation in a sample would be useful for a wide range of clinical and research applications.
Accurate measures of data obtained from mRNA, such as gene expression, depend on the state of the mRNA sample being qualified. Generally, the degradation state of an mRNA population can be assessed by using a single mRNA species as a surrogate molecule. Using two Taqman assays designed to target this mRNA at the 3′- and the 5′-end of the molecule, the mRNA population can be converted to cDNA either through random priming or oligo(dT) priming and the cDNA population can be interrogated simultaneously with these two assay. The linked:unlinked ratio between the two assays can then be used to provide an index of degradation state. The reliability of this assay however, depends on the processivity of the reverse transcriptase utilized, which can convolute any indices of degradation state.